Summary Our goal is to develop and commercialize a multiplexed assay for dominant hereditary ataxias to rapidly and accurately analyze short tandem repeat (STR) lengths leading to a positive impact on DNA-based diagnostics screening. Ataxia is the loss of full control of body movements and indicates a dysfunction of the nervous system and have a range of causes including hereditary and acquired. Spinocerebellar ataxia (SCA) is a rare, dominantly inherited disease family with over 40 known types. The most common SCAs (1, 2, 3, 6 and 7) are caused by STR expansions in ATXN1, ATXN2, ATXN3, CACNA1a, and ATXN7, respectively and responsible for >60% cases worldwide. In the US, there are an estimated 150,000 people affected with hereditary ataxia. Due to the overlapping nature of disease presentation, symptoms, and age of onset between different ataxias, molecular profiling is key to diagnosis, however undiagnosed and misdiagnosed estimates are between one third and one fifth of the potential patients due incomplete or inaccurate testing. High-quality allele sizing is critical for accurate diagnosis. Next-generation sequencing panels are available although unsuitable for STR expansions due to limitations in read size and sequence complexity. Other methods, including single assay PCR followed by Southern blot, are cumbersome, costly, and difficult to size accurately. Our approach utilizes PCR AmplideX chemistry combined with capillary electrophoresis (CE) to provide a fast, accurate and cost-effective multiplex test compared to currently available assays. We believe a unified testing paradigm will advance clinical research, molecular diagnostics and drug development for the most prevalent dominant ataxias. To support a rapid adoption of the kit, a single-tube multiplex PCR test will be developed. CE instruments are efficient, practical, and are broadly used in most molecular diagnostics laboratories. To improve robust early assay design, the Oxford Nanopore (NP) 3rd generation sequencing platform will be used as a reference assay for determining concordance as an independent orthogonal method. Aim 1: Enable the detection of each of the disease-causing expanded repeat regions of ATXN1, ATXN2, ATXN3, CACNA1a, and ATXN7 individually using AmplideX PCR/CE chemistry. Aim 2: Develop a unified multiplex PCR-based assay to determine STR sizing for ATXN1, ATXN2, ATXN3, CACNA1a, and ATXN7 in a single tube on ?50 clinical isolates. Our approach will provide a fast, accurate and cost-effective multiplex test compared to available assays, advancing clinical research, molecular diagnostics and drug development for dominant ataxias. Our approach will result in an on-market PCR/CE RUO kit within two years after funding support.